Stochastic Gene Expression in Single Gene Oscillator Variants

It is infeasible to understand all dynamics in cell, but we can aim to understand the impact of design choices under our control. Here we consider a single gene oscillator as a case study to understand the influence of DNA copy number and repressor choice on the resulting dynamics. We first switch the repressor in the oscillator from the originally published lacI to treRL, a chimeric repressor with a lacI DNA binding domain that is inducible by trehalose. This slightly modified system produces faster and more regular oscillations than the original lacI oscillator. We then compare the treRL oscillator at three different DNA copy numbers. The period and amplitude of oscillations increases as the copy number is decreased. We cannot explain the change in period with differential equation models without changing delays or degradation rates. The correlation and phase coherence between daughter cells after cell division also tend to fall off faster for the lower copy oscillator variants. These results suggest that lower copy number variants of our single gene oscillator produce more synchronized oscillations

Characterization of the Active Site and Insight into the Binding Mode of the Anti-angiogenesis Agent Fumagillin to the Manganese(II)-Loaded Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g≈2 EPR signal of mononuclear Mn(II) with A av(55Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms. Other features in the spectrum were observed only as the temperature was raised from that of liquid helium. The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 ... 5 non-Kramer’s doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state. This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers’ spin ladder. Upon the addition of the anti-angiogenesis agent fumagillin to [MnMn(EcMetAP-I)], very small changes were observed in the EPR spectrum. MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I. Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions

Divalent Metal Binding Properties of the Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 ± 0.2 μM and 0.2 ± 0.2 μM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (\u3e50 equiv) resulted in the loss of ∼50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 ± 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion (∼5%) of the sample exhibited detectable spin−spin interactions at pH values \u3e 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. 1H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H2O and D2O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed

Analyzing the Catalytic Role of Asp97 in the Methionine Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co(II) d–d spectra for wild-type, D97E and D97A EcMetAP-I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) EcMetAP-I, and only a doubling of intensity was observed upon addition of a second Co(II) ion. In contrast, the electronic absorption spectra of [Co_(D97N EcMetAP-I)] and [CoCo(D97N EcMetAP-I)] were distinct, as were the EPR spectra. On the basis of the observed molar absorptivities, the Co(II) ions binding to the D97E, D97A and D97N EcMetAP-I active sites are pentacoordinate. Combination of these data suggests that mutating the only nonbridging ligand in the second divalent metal-binding site in MetAPs to an alanine, which effectively removes the ability of the enzyme to form a dinuclear site, provides a MetAP enzyme that retains catalytic activity, albeit at extremely low levels. Although mononuclear MetAPs are active, the physiologically relevant form of the enzyme is probably dinuclear, given that the majority of the data reported to date are consistent with weak cooperative binding

Cyst fluid antibiotic concentrations in polycystic kidney disease: Differences between proximal and distal cysts

Cyst fluid antibiotic concentrations in polycystic kidney disease: Differences between proximal and distal cysts. The concentrations of several antibiotics were measured in the cyst fluid of six adult patients with polycystic kidney disease Seventy-nine cysts were aspirated at surgery or autopsy Sixty-one cysts could be categorized as arising from the proximal nephron and 16 from the distal nephron by cyst fluid to serum sodium ratios. Serum, urine, and cyst fluid were simultaneously analyzed for sodium, creatinine, and various antibiotics. Gentamicin, tobramycin, cephapirin, and ticarcillin were either undetectable or present in low concentrations in renal cysts. Cyst fluid antibiotic concentrations did not correlate with cyst volume or creatinine clearance. Cysts of proximal nephron origin had higher antibiotic concentrations than distal cysts. In one patient with normal renal function, inulin was undetectable in renal cysts after a continuous 36-hour i.v. infusion. Para-aminohippurate, however, was detected in the renal cysts of this patient. These data help explain the poor clinical response of infected renal cysts to antibiotic therapy. They also suggest that antibiotics and other solutes may enter cyst fluid across tubular cells in addition to entry by glomerular filtration.Concentration intra kystique d'antibiotiques dans la maladie poly kystique rénale: Différences entre les kystes proximaux et distaux. Les concentrations de plusieurs antibiotiques dans le liquide des kystes ont été mesurées chez six sujets adultes atteints de maladie polykystique. Soixante dix neuf kystes ont été ponctionnés pendant des interventions chirurgicales ou des autopsies. Soixante et un kystes ont pu être classés comme proximaux et seize comme distaux en fonction du rapport de concentration de sodium kyste/plasma. Des déterminations de concentration de sodium, de creatinine et de divers antibiotiques ont été réalisées simultanément pour le plasma, l'urine et le liquide des kystes. La gentamicine, la tobramycine, la cephapirine et la ticarcilline étaient soit non détectables soit à des concentrations très faibles dans les kystes. Les concentrations d'antibiotiques dans les kystes n'étaient pas corrélées avec le volume du kyste ou la clearance de la creatinine. Les kystes proximaux avaient des concentrations d'antibiotiques plus élevées que les kystes distaux. Chez un malade dont la fonction rénale était normale l'inuline n'était pas détectable dans les kystes après une perfusion continue de 36 heures. Le para-aminohippurate, cependant, a été détecté dans les mêmes kystes. Ces résultats permettent de comprendre la réponse clinique faible des kystes infectés au traitement antibiotique. Ils suggèrent aussi que les antibiotiques ainsi que d'autres substances dissoutes peuvent pénétrer dans les kystes à travers les cellules tubulaires en sus de la pénétration par filtration glomérulaire

Structurally Distinct Active Sites in the Copper(II)-Substituted Aminopeptidases from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e and \u3cem\u3eEscherichia coli\u3c/em\u3e

The aminopeptidase from Aeromonas proteolytica (AAP) was titrated with copper, which bound sequentially at two distinct sites. Both the mono- and disubstituted forms of AAP exhibited catalytic hyperactivity relative to the native dizinc enzyme. Monosubstituted AAP exhibited an axial Cu(II) EPR spectrum with slight pH dependence:  at pH 6.0 g|| = 2.249, g⊥ = 2.055, and A||(63/65Cu) = 1.77 × 10-2 cm-1, whereas at pH 9.65 g|| = 2.245, g⊥ = 2.056, and A||(63/65Cu) = 1.77 × 10-2 cm-1. These data indicate oxygen and nitrogen ligation of Cu. AAP further substituted with copper exhibited a complex signal with features around g ∼ 2 and 4. The features at g ∼ 4 were relatively weak in the B0 ⊥ B1 (perpendicular) mode EPR spectrum but were intense in the B0 || B1 (parallel) mode spectrum. The g ∼ 2 region of the perpendicular mode spectrum exhibited two components, one corresponding to mononuclear Cu(II) with g|| = 2.218, g⊥ = 2.023, and A||(63/65Cu) = 1.55 × 10-2 cm-1 and likely due to adventitious binding of Cu(II) to a site distant from the active site. Excellent simulations were obtained for the second component of the spectrum assuming that two Cu(II) ions experience dipolar coupling corresponding to an inter-copper distance of 5 Å with the two Cu(II) gz directions parallel to each other and at an angle of ∼17° to the inter-copper vector (ℋ = βB·gCuA·SCuA + βB·gCuB·SCuB + [S·A·I]CuA + [S·A·I]CuB + [SCuA·J·SCuB]; g||(CuA,CuB) = 2.218, g⊥(CuA,CuB) = 2.060; A||(CuA,CuB)(63/65Cu) = 1.59 × 10-2 cm-1, Jisotropic = 50 cm-1, rCu-Cu = 4.93 Å, and χ = 17°). The exchange coupling between the two copper ions was found to be ferromagnetic as the signals exhibited Curie law temperature dependence. The Cu−Cu distance of ∼5 Å indicated by EPR was significantly higher than the inter-zinc distance of 3.5 Å in the native enzyme, and the dicopper species therefore represents a novel dinuclear site capable of catalysis of hydrolysis. In contrast to AAP, the related methionyl aminopeptidase from Escherichia coli (EcMetAP) was found to bind only one Cu(II) ion despite possessing a dinuclear binding site motif. A further difference was the marked pH dependence of the signal in EcMetAP, suggestive of a change in ligation. The structural motifs of these two Cu(II)-substituted aminopeptidases provide important insight into the observed catalytic activity

Investigation of OSB Thickness-Swell Based on a 3-D Density Distribution. Part II. Variations in Thickness-Swell and Internal Stresses

A recently developed finite element (FE) model was used to examine the thickness-swell, density changes, and internal stresses in oriented strandboard (OSB) panels under moisture loading. The model accounts for the nonlinear mechanical behavior of OSB and for the moisture transport through the specimen. The FE model is based on the 3-D density variation of the board. The density variation, resulting from manufacturing processes, affects the uniformity of thickness-swell in OSB and is often exacerbated by continuous sorption of moisture, which leads to potentially damaging internal stresses in the panel. The model was validated through comparison of experimental results. The use of the model is illustrated by quantifying the effects of resin content changes on thickness swell, and the examination of internal stresses and bond failures in an OSB specimen

Synthesis, Molecular Structure, and Reactivity of Dinuclear Copper(II) Complexes with Carboxylate-Rich Coordination Environments

The dinucleating ligand N,N‘-(2-hydroxy-5-methyl-1,3-xylylene)bis(N-(carboxymethyl)glycine) (CH3HXTA) has been used to synthesize the dinuclear Cu(II) bis(pyridine) complex Na[Cu2(CH3HXTA)(Py)2]·1.5(1,4-dioxane) (Na(1)):  triclinic space group P1̄ (a = 12.550(3) Å, b = 13.413(3) Å, c = 13.540(4) Å, α = 117.12(2)°, β = 104.70 (2)°, and γ = 92.13(2)°). The structure shows two distinct distorted square pyramidal Cu(II) centers with each Cu(II) ion bound by two carboxylate oxygen atoms, one amine nitrogen atom, a phenolate oxygen atom, and one pyridine nitrogen atom. The Cu--Cu separation is 3.531 Å, and the Cu1−O1−Cu2 angle is 123.7°. The phenyl ring of the CH3HXTA ligand is twisted relative to the Cu1−O1−Cu2 plane, and the resulting dihedral angle is 44.2°. The electronic absorption spectrum of 1 in aqueous solution at pH 3 suggests a shift toward trigonal bipyramidal Cu(II) coordination in solution. Spectral titration of Na[Cu2(CH3HXTA)(H2O)2] with L (where L = pyridine or sodium cyanide) results in complexes with terminal L groups. These exogenous ligands appear to bind in a positive cooperative stepwise fashion. Variable-temperature magnetic susceptibility data for 1 indicate that the Cu(II) ions are antiferromagnetically coupled (−2J = 168 cm-1). X-band EPR spectra of an aqueous solution of 1 shows isotropic signals with g = 2.14, while a powdered sample of 1 provides no EPR spectrum. A ΔMs = 2 transition at g = ∼4.5, expected for weakly magnetically coupled Cu(II) ions, is not observed for powdered samples but is observed for a methanolic solution sample of 1. On the basis of these data, the two Cu(II) ions are antiferromagnetically coupled in the solid state but due to a coordination geometry change become weakly ferromagnetically or antiferromagnetically coupled in solution. 1H NMR studies on a methanol solution of 1 are consistent with weak spin-coupling in solution